Integrins are heterodimeric receptors that mediate a wide variety of important interactions both between cells and between cells and the extracellular matrix via ligand binding. All integrins have an α subunit and a β subunit. Within the α subunit a region referred to as the A domain (or I domain) is known to be an important mediator of ligand binding. A similar region, the A-like domain, is present in many β subunits. These heterodimeric αβ integrins contain a von Willebrand Factor (vWF) A domain, βA, in their β subunits; and nine integrins also contain a second such domain, αA, in their α subunits. Many integrins are thought to exist in two conformations, a low affinity state (the “closed” or “unliganded” conformation”) and a high affinity state (the “open” or “liganded” conformation), the latter of which is responsible for high affinity ligand binding.
Integrins transduce signals that mediate the effects of the matrix on the physiological activity of cells (e.g., motility, proliferation, differentiation). Moreover, integrins play a role in inflammation and in oncogenic cell transformation, metastasis, and apoptosis. Thus, there is considerable interest in identifying compounds that can activate or inhibit the activity of one or more integrins.
In order for an efficient integrin-ligand binding to occur, it is thought that the integrin must be in its high affinity configuration. It appears that inside-out signals generated when cells are activated by a variety of stimuli apparently switch integrins from a low affinity state to a high affinity state. This functional upregulation is associated with conformational changes in the extracellular regions of integrins that include the A domain of the α subunit and the A-like domain of the β subunit (Smith et al. 1988 J. Biol. Chem. 263:18726).
The integrin A-domain assumes a dinucleotide-binding fold (Lee et al. 1995 Cell 80:631; Emsley et al. 1997 J. Biol. Chem. 272:28512; Li et al. 1998 J. Cell Biol. 143:1523; Nolte et al. 1999 FEBS Lett. 452:379; and Legge et al. 2000 J. Mol. Biol. 295:1251), with a metal ion dependent adhesion site (MIDAS) on its top, and is connected through a C-terminal α7 helix at its bottom to the body of the integrin. MIDAS and its surrounding exposed side-chains form the binding site for physiologic ligands (Li et al., supra; Michishita et al. 1993 Cell 72:857-867; Kamata et al. 1994 J. Biol. Chem. 269:26006-26010; Kern et al. 1994 J. Biol. Chem. 269:22811-6; Edwards et al. 1998 J. Biol. Chem. 273:28937-44; Zhang et al. 1999 Biochemistry 38:8064-71) and certain antagonists (Rieu et al. 1996 J. Biol. Chem. 271:15858-15861). In the “open” conformation, three non-charged resides in the protein directly coordinate the metal ion in MIDAS, a pseudoligand or ligand glutamate residue (Lee et al., supra; Li et al., supra; Emsley et al. 2000 Cell 100:47-56) completes metal coordination. In the “closed” form, the amphipathic C-terminal α7 helix is shifted upwards by 10 Å compared to the “open” form, wrapping around the rest of the domain. This large shift is associated with a change in metal coordination, where one of the three coordinating residues, a threonine, is now replaced with an aspartate, and a water molecule replaces the glutamate in completing the metal ion coordination sphere (Lee et al. 1995 Structure 3:1333-1340). These changes in metal coordination and topology of MIDAS are similar to those described in the structurally homologous G proteins (Lee et al., supra).
The crystal structure of four integrin A-domains (CD11b, CD11a, CD49a and CD49b) have been reported to date (Lee et al., supra; Lee et al. 1995 Structure 3:1333; Emsley et al., supra; Li et al., supra; Emsley et al. 1997 J. Biol. Chem. 272:28512). All, with the exception of integrin CD11b A-domain (11bA), were found only in the “closed” form, leading to the suggestion that the “open” form is a non-informative crystal artifact (Baldwin et al. 1998 Structure 6:923-935). Three studies support the view that the “open” form of the integrin A-domain equates with the “high” affinity state (Li et al. 1998 J. Cell Biol. 143:1523; Rieu et al., supra; Oxvig et al. 1999 Proc. Nat'l Acad. Sci. USA 96:2215-20). In the first, point mutations in CD11bA that are predicted on structural grounds to destabilize the “closed” structure, increased the proportion of the “high affinity” form in solution (Li et al. 1998 J. Cell Biol. 143:1523). In the second, the binding site for an “activation-dependent” monoclonal antibody mapped to a conformationally sensitive region of the A-domain (Oxvig et al., supra). The third study showed that an A-domain in complex with a short collagen peptide assumed the “open” conformation, and suggested that the “open” form can only be obtained in the presence of ligand (Emsley et al., supra). While it has been suggested that the ligand causes the conformational change in integrins, at least one study suggests that integrins can exist in high affinity state even in the absence of ligand (Smith and Cheresh 1988 J. Biol. Chem. 263:18726-31). In addition, several studies suggested that ligand binding affinity in heterodimeric integrins can be altered in an allosteric manner (Li et al. 1998 J. Cell Biol. 143:1523; Edwards et al. 1998 J. Biol. Chem. 273:28937-28944; Calderwood et al. 1998 J. Biol. Chem. 273:5625; Zhang et al. 1996 J. Biol. Chem. 271: 29953-7).